Use of nitrites and nitrates and compositions containing these

ABSTRACT

Inorganic anions nitrate and nitrite influence metabolic rate and glucose homeostasis. Infusion of nitrite iv caused an acute drop in resting energy expenditure (oxygen consumption) and nitrate, when given perorally, caused a drop in oxygen consumption during exercise and a depression of the increase in blood glucose observed after an oral glucose tolerance test. The doses of nitrate and nitrite did not cause any detectable change in methemoglobin levels of blood. Also, nitrate and nitrite did not alter lactate levels in blood. This discovery provides useful treatments to regulate the energy expenditure and glucose homeostasis of a mammal by administration of inorganic nitrite and/or nitrate.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Phase patent application of PCT/SE2008/050212,filed Feb. 26, 2008, which claims priority to Swedish patent applicationSerial No. 0700520-0, filed Feb. 26, 2007, and U.S. Provisional patentapplication Ser. No. 60/919,709, filed Mar. 22, 2007, all of which arehereby incorporated by reference in the present disclosure in theirentirety.

TECHNICAL FIELD

This invention relates to the field of medicine and pharmaceuticals, inparticular pharmaceuticals and therapeutic methods for loweringmetabolic rate, oxygen consumption and/or glucose homeostasis in a humanpatient or another mammal, based on the administration of nitratesand/or nitrites to said patient or mammal.

BACKGROUND ART

Nitrate (NO₃ ⁻ ) and nitrite (NO₂ ⁻ ) are generally viewed as unwantedresidues in the food chain with potentially harmful effects (JointFAO/WHOExpert Committee on Food Additives (JECFA). Safety Evaluation ofCertain Food Additives. WHO, 1970. ISBN 9241660503; TANNENBAUM, S. R.,et al. Nitrite in human saliva. Its possible relationship to nitrosamineformation. J cancer Ins. 1974, vol. 53, p. 79-84; BARTSCH, H., et al.Inhibitors of endogenous nitrosation: mechanisms and implications inhuman cancer prevention. Mutation Res. 1988, vol. 202, p. 307-324).Proposed harmful effects of these anions include promotion of gastriccancers and other malignancies and development of methemoglobinemia ininfants. Because of this the levels of nitrate/nitrite are strictlyregulated in food and drinking water.

Recent studies indicate that nitrate and nitrite can have significantbiological effects in the body and that these effects may be beneficial(LUNDBERG, Jon O., et al. Nitrate, becteria and human health. Nat RevMicrobiol. 2004, no. 2, p. 593-602). For example the nitrite anion cancause vasodilatation at near physiological concentrations when tested invitro (MODIN, A., et al. Nitrite-derived nitric oxide: a possiblemediator of ‘acidic-metabolic’ vasodilation. Acta Physiol Scand. 2001,vol. 171, p. 9-16) or when infused intra-arterially to humans (COSBY,K., et al. Nitrite reduction to nitric oxide by deoxyhemoglobinvasodilates the human circulation. Nat Med. 2003, no. 9, p. 1498-505).Nitrate can be converted to nitrite in vivo in a process dependent oncommensal bacteria (SPIEGELHALDER, B., et al. Influence of dietarynitrate on nitrite content of human saliva: possible relevance to invivo formation of N-nitroso compounds. Food Cosmet Toxicol. 1976, no.14, p. 545-548). When nitrate is ingested it is rapidly absorbed intoblood and then accumulates in saliva. In the oral cavity bacteria reduceparts of the dietary nitrate to nitrite and nitrite can then enter thesystemic circulation. (LUNDBERG, Jon O., et al. Inorganic nitrate is apossible source for systemic generation of nitric oxide. Free Radic BiolMed. 2004, vol. 37, p. 395-400).

To date the focus among researchers has been on the cardiovasculareffects of nitrite after its in vivo reduction to the vasodilator nitricoxide (NO) (COSBY et al. (supra); DURANSKI, M. R., et al. Cytoprotectiveeffects of nitrite during in vivo ischemia-reperfusion of the heart andliver. J Clin Invest. 2005, vol. 115, p. 1232-1240; GLADWIN, M. T., etal. The emerging biology of the nitrite anion. Nat Chem Biol. 2005, no.1, p. 308-14; LARSEN, F. J., et al. Effects of dietary nitrate on bloodpressure in healthy volunteers. N Engl J Med. 2006, vol. 355, p.2792-3).

WO 2005/004884 A (US GOVERNMENT ET AL.) 2005 Jan. 20 and WO 2005/007173A (US GOVERNMENT ET AL.) 2005 Jan. 27 describe a method to administer anitrite salt specifically to obtain vasodilatation in a subject. Noeffects of low-dose nitrate/nitrite on energy expenditure or glucosehomeostasis have been described.

SUMMARY OF THE INVENTION

The inventors have surprisingly shown that the metabolic rate and/or theoxygen consumption can be influenced in a mammal (locally in isolatedtissues or organs or systemically in the whole body), by administeringinorganic nitrite (NO₂ ⁻ ) and/or nitrate (NO₃ ⁻ ) to said mammal in anamount of nitrite and/or nitrate sufficient to decrease oxygenconsumption. The oxygen consumption is decreased without causingsignificant hypotension and does not cause any significant increase ofthe methemoglobin level in said mammal.

The invention also makes available a method for lowering the metabolicrate in a mammal, wherein a pharmaceutical composition comprisinginorganic nitrite (NO₂ ⁻ ) and nitrate (NO₃ ⁻ ) is administered to saidmammal in an amount of nitrite and nitrate which is sufficient todecrease oxygen consumption. The oxygen consumption is decreased withoutcausing significant hypotension.

In particular, the invention makes available a method for lowering themetabolic rate in a mammal, wherein a pharmaceutical compositioncomprising inorganic nitrite (NO₂ ⁻ ) is administered to said mammal inan amount of nitrite which is sufficient to decrease oxygen consumption.The oxygen consumption is decreased without causing significanthypotension. The nitrite can be administered perorally or parenterally.When administered parenterally, said nitrite can be administeredintravenously at a dose of about 0.01 to about 10 000 nmoles/kg/min.

The invention also makes available a method for lowering the metabolicrate in a mammal, wherein a pharmaceutical composition comprisinginorganic nitrate (NO₃ ⁻ ) is administered to said mammal in an amountof nitrate which is sufficient to decrease oxygen consumption, but whichdoes not increase methemoglobin levels in said mammal, wherein saidnitrate is administered perorally in the form of a nitrate salt at adose of about 0.01 to about 100 mmol/kg/24 h. When given perorally,nitrate can be seen as a precursor of nitrite.

The invention also makes available a composition, use and method whereininorganic nitrite and/or nitrate is/are combined with polyphenols.

The influence on, or regulation of, metabolic rate and/or oxygenconsumption can be used as a step in the treatment, prevention oramelioration of a pathological or physiological condition wheremetabolic stress is a distinctive feature. Such stress may be present ine.g. intensive care patients, patients undergoing surgery, patients withmalnutrition, patients with cancer and anorexia, patients with burninjury, trauma patients, neonates and prematures, patients with anorexianervosa, patients scheduled for solid organ transplantation or patientshaving undergone an solid organ transplantation. In addition, a decreasein oxygen consumption with a lower need for oxygen in the tissues, isdesirable in any pathological situation characterized by low oxygenavailability, including; chronic obstructive pulmonary disease (COPD),inflammatory airway disease such as asthma, pulmonary hypertension,congestive heart disease, interstitial lung disease, pulmonary embolism,ischemic heart disease, peripheral artery disease; sleep apnea syndrome.

The inventive findings can also be applied to the regulation of themetabolic rate as a step in treating, preventing or ameliorating acondition of disturbed glucose homeostasis (glucose control) including:diabetes mellitus type 1 and type 2, prediabetes, intensive carepatients, surgical trauma, metabolic syndrome, obesity, burn injury,drug-induced diabetes. The term “glucose control” is used in itsbroadest sense, as it is known that the stabilization of blood glucoselevels is important for many reasons and concerns many patient groups,as well as healthy subjects. It is general knowledge that diabeticpatients are susceptible to complications such as neuropathies,cardiomyopathy, vascular disease, poor wound healing and blindness. Evenpatients suffering with simple hypoglycaemia would benefit from glucosecontrol. Further it is hypothesised that many common health conditionsother than diabetes have a component of insufficient or disturbedglucose control. For example obesitas is closely associated with glucoseand insulin. It is also suggested that emotional problems such asconcentration difficulties and mood swings are associated with poorglucose control. Due to the well documented adverse effects ofhyperglycaemia as well as hypoglycemia, it is important to maintainproper glucose control in both diabetic and non-diabeticpatients/subjects.

The invention also provides methods for treatment, alleviation and/orprevention of clinical conditions, comprising administering an effectiveamount of a nitrate and/or a nitrite to a patient in need thereofsufficient to treat, alleviate and/or prevent such condition.

As a consequence of the present findings and the conclusions reached bythe inventors, the present invention also makes available new uses ofnitrites and/or nitrates, for the manufacture of pharmaceuticalproducts, enteral or parenteral nutritional solutions, or nutritionalsupplements, for administration to both healthy persons, such asathletes, or to patients, suffering from one or more of the conditionsexemplified in the description.

Further embodiments will become evident to the skilled person upon studyof the figures, description and examples, as well as the appendedclaims, incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in closer detail in the followingdescription, non-limiting examples and claims, with reference to theattached drawings in which:

FIG. 1 shows a graph illustrating numerous ways in which the combinationof nitrate and polyphenols synergistically act to increase thebioavailability of nitric oxide and at the same time to reduce theformation of harmful compounds such as oxygen radicals and nitrosamines.For detailed explanation see text.

FIG. 2 is a graph showing changes in oxygen consumption (VO₂) followingiv infusion of sodium nitrite in increasing doses. Nitrite was infusedover a 10 min period in non-smoking healthy male volunteers (30-70years).

FIG. 3 is a graph showing the effects of a dietary supplementation withsodium nitrate or sodium chloride (placebo) on plasma concentrations ofnitrite measured at rest and immediately after exercise in 9 healthymale volunteers.

FIG. 4 is a bar diagram showing the oxygen consumption (VO₂) and heartrate (HR) measured at 6 different work rates after a 3-day dietarysupplementation with sodium nitrate (0.1 mmol/kg/min, NIT) or an equalamount of sodium chloride (CON). The study had a randomized double-blindcross-over design with a washout period of at least 10 days between thetests. *p<0.05, **p<0.01.

FIG. 5 is a graph showing oxygen consumption during bicycle exercise at80% of VO_(2peak) in 9 healthy male volunteers. Measurements were madeafter a 3-day dietary supplementation with sodium nitrate (0.1mmol/kg/day) or an equal amount of sodium chloride (placebo). Thedifference between nitrate and placebo periods was significant (p<0.01).

FIG. 6 is a bar diagram showing plasma lactate concentration measured at6 different work rates after dietary supplementation with sodium nitrate(0.1 mmol/kg/day for 3 days, filled bars) or an equal amount of sodiumchloride (placebo, empty bars).

FIG. 7 consists of three graphs, showing changes in blood glucose levelsafter an oral challenge with glucose for three test subjects in adouble-blind, placebo-controlled cross-over study (FIGS. 7a, 7b and 7c).A standard oral glucose tolerance test was performed. The subjects(healthy non-smoking volunteers) had their diet supplemented for 3 dayswith either sodium chloride (placebo) or sodium nitrate (Nitrate) at adose of 0.1 mmol/kg/day.

FIG. 8 is a graph showing changes in blood glucose levels after an oralchallenge with glucose in 8 additional subjects in a double-blind,placebo-controlled cross-over study. A standard oral glucose tolerancetest was performed. The subjects (healthy non-smoking volunteers) hadtheir diet supplemented for 3 days with either sodium chloride (PLACEBO)or sodium nitrate (NITRATE) at a dose of 0.1 mmol/kg/day. Data arepresented as mean±SEM.

FIG. 9 is a graph showing the effect of a two-week intervention withbeetroot juice (fresh juice 3-4 dl/day) on systolic, diastolic and meanarterial (MAP) blood pressure in a 43 year old male with hypertension.

FIG. 10 shows the plasma nitrate and nitrite concentrations afterintravenous infusion of nitrate. a) shows plasma nitrate concentrations,b) shows plasma nitrite concentrations and c) shows plasma nitriteconcentrations in wild type (C57BL/6), germ free and knockout (eNOS)mice.

FIG. 11 is a graph showing enhanced post-ischemic blood flow afternitrate infusion.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present method and compositions are described in the form ofembodiments thereof, it is to be understood that this invention is notlimited to the particular configurations, method steps, and materialsdisclosed herein as such configurations, steps and materials may varysomewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

It must also be noted that, as used in this specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

The term “about” when used in the context of numeric values denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. Said interval can be +/−2% of the given value, preferably +/−5%,and most preferably +/−10% of the numeric values, where applicable.

The term “mammal” is intended to encompass all mammals, and inparticular humans, pets and agriculturally significant animals, as wellas animals used in competitions, such as horses and dogs.

The term “significant hypotension” means in this context an acutereduction of systolic and/or diastolic blood pressure, accompanied byclinical symptoms of hypotension such as dizziness, nausea, pallor, lossof consciousness, etc. Said symptoms may occur in various degrees, andit is preferred that they are entirely avoided, minimized or eliminatedas far as possible, or at least to an extent that they are clinicallyinsignificant.

The term “metabolic syndrome” is here defined as a combination ofmedical disorders that increase the risk for cardiovascular disease anddiabetes in a human. Symptoms and feature include fastinghyperglycaemia, diabetes mellitus type 2 or impaired fasting glucose,impaired glucose tolerance or insulin resistance, high blood pressure,central obesity, decreased HDL cholesterol, elevated triglycerides andelevated uric acid levels.

The term “insulin resistance” is here defined as a condition in whichnormal amounts of insulin are inadequate to produce a normal insulinresponse from fat, muscle and liver cells.

The term “metabolism” is used to define the complete set of chemicalreactions that occur in living cells and “metabolic rate” is defined asthe speed of metabolism of a mammal. The term “energy expenditure” ishere defined as the amount of energy expended for a certain metabolicrate.

The term “oxygen consumption” is defined as the amount of oxygen (O₂)consumed by a mammal and is usually expressed as ml of pure oxygenconsumed/min. “Oxygen consumption” relates to the amount of oxygenconsumed by a mammal as whole but also to oxygen consumption in anisolated tissue or organ, such as, but not limited to, heart, liverbrain or other tissue exposed to ischemia.

Methemoglobin is a form of hemoglobin in which the iron in the hemegroup is in the Fe³⁺ state, not the Fe²⁺ of normal hemoglobin.Methemoglobin is unable to carry oxygen. Methemoglobinemia is defined asa blood disorder characterized by the presence of a higher than normallevel of methemoglobin in the blood.

The term “catabolism” is defined as the metabolic process that breaksdown molecules into smaller units. It is made up of degradative chemicalreactions in the living cell.

The term “functional food” relates to any fresh or processed foodclaimed to have a health-promoting and/or disease-preventing propertybeyond the basic nutritional function of supplying nutrients. Functionalfoods are sometimes called nutraceuticals. The general category includesprocessed food made from functional food ingredients, or fortified withhealth-promoting additives, like “vitamin-enriched” products, and also,fresh foods (eg vegetables) that have specific claims attached.Fermented foods with live cultures are often also considered to befunctional foods with probiotic benefits.

The inventors have surprisingly shown that the metabolic rate and/or theoxygen consumption can be influenced in a mammal (locally orsystemically), by administering inorganic nitrite (NO₂ ⁻ ) and/ornitrate (NO₃ ⁻ ) to said mammal in an amount of nitrite and/or nitratesufficient to decrease oxygen consumption. The decreased oxygenconsumption is achieved without causing significant hypotension andwithout causing any significant increase of the methemoglobin level insaid mammal. In case of local reduction of the metabolic rate, theoxygen consumption is decreased in an isolated tissue or organ such asthe heart, liver, brain or other tissue that is exposed to ischemia (acondition in which blood flow, and thus oxygen, is restricted to a partof the body). In such cases the interaction of reaction products(including NO) of nitrite and/or nitrate with enzymes of themitochondrial respiratory chain and subsequent inhibition of respirationleads to lowering of oxygen demand which is beneficial for an ischemictissue. This effect resembles hibernation. Because the generation ofactive nitrite and/or nitrate reaction products are maximized inischemic tissues the effect of oxygen consumption will be mostpronounced at these sites. In one particular embodiment, the oxygenconsumption is lowered in the heart.

The surprising finding that nitrite and its precursor nitrate affectssuch vital physiological processes as metabolic rate and/or oxygenconsumption can be used therapeutically, e.g. in prophylaxis,alleviation or treatment of several conditions. In an attempt toincrease systemic nitrite levels, nitrite and/or nitrate can be given byenteral administration (orally, in the form of a liquid, semi-solid orsolid preparation, such as a chewing gum, tablet, lozenge, wafer, cake,bar or the like) or by parenteral administration (intravenous,transdermal, transcutaneous, by inhalation, rectally, vaginally,topical, intraperitoneally, intra muscular, subcutaneous, sublingual orany other way of parenteral administration). The nitrite and/or nitratecomprising composition and possible further combinations describedherein can be administered continuously or as single bolus doses.

In principle, also other additional approaches can lead to increasedsystemic or local nitrite levels. The most obvious is to administernitrite or its precursor nitrate as such, but this may be supplementedby or enhanced by increasing the gastric pH (e.g. with an acidsuppressive drug such as a proton pump inhibitor or H₂ receptorantagonist or antacida) to maximise nitrite survival in the stomach andthereby the systemic delivery of nitrite. Alternatively, theadministration of nitrite or nitrate can be supplemented by or enhancedby interfering with the oral microflora in order to maximise the numberof nitrate reducing species. This can be achieved through the deliveryof “probiotic” nitrate reducing bacteria or selective treatment with anantibiotic to favour the nitrate reducing species.

It is likely that an optimal dose-interval exists, meaning that below acertain plasma level of nitrite the effects are insufficient andcorrespondingly, that over a certain level the effect is lower, andpossibly accompanied by side effects. Using intravenous administration,the nitrite is preferably administered in a dose within the interval ofabout 0.01 to about 10,000 nmoles/kg/min, preferably about 0.01 to about1000 nmoles/kg/min, more preferably about 0.1 to about 100nmoles/kg/min, most preferably about 1 to about 20 nmoles/kg/min. It ispresently contemplated that the most preferred dose is less than about15 nmoles/kg/min. For comparison, it should be noted that the nitritedose used in the treatment of cyanide poisoning is about 100 000nmoles/kg, or about 300-400 mg given as a single dose. The nitrite canalso be administered as one or more bolus doses, preferably 0.01-100μmoles/kg body weight, more preferably 0.1-10 μmoles/kg body weight, andeven more preferably 0.1-2 μmoles/kg body weight.

For the use of a nitrate salt perorally, a dose of about 0.01-100mmol/kg/24 h is currently preferred or more preferably a dose of about0.01-10 mmol/kg/24 h, even more preferably 0.1-1 mmol/kg/24 h.

Importantly, the administered dose of nitrate or nitrite should notinduce production of more than about 10% methemoglobin, preferably notmore than about 5% methemoglobin, and more preferably not more thanabout 2% methemoglobin. Most preferred is that the dose of nitrate ornitrite does not induce any measurable change in methemoglobin in thesubject when administered or ingested according to the prescribed dose.

Combinations of nitrate and nitrite salts can also be used. According toone embodiment, nitrate and nitrite are given orally in a dose ratiointerval of about 5:1 to about 100:1 (nitrate:nitrite), such as 5:1,10:1, 30:1, 50:1, 70:1 and 100:1. Preferably the dose ratio is about10:1. This will ensure acute effects of the nitrite as soon as it isabsorbed, and then provide a sustained effect of the nitrate followingits bioconversion into nitrite.

In one embodiment the composition comprising inorganic nitrite and/ornitrate is a pharmaceutical composition comprising inorganic nitriteand/or nitrate in an amount which is sufficient to decrease oxygenconsumption, but which does not increase the methemoglobin level in asubject when administered to said subject in a prescribed dose.Optionally the composition comprises another pharmaceutically activecompound.

Alternatively, the composition comprising nitrite and/or nitrate is anutritional supplement, an enteral nutritional solution, an infantformula, a snack product or a parenteral nutritional solution comprisinginorganic nitrite and/or nitrate in an amount which is sufficient todecrease oxygen consumption, but which does not cause significanthypotension in a subject when ingested by said subject in a prescribeddose.

Pharmaceutically acceptable salts of nitrate and nitrite include but arenot limited to sodium, potassium, calcium, zinc, arginine and ammonium.Sodium and potassium salts are presently most preferred. The nitrite andnitrate salts may be of synthetic origin, but may also be isolated fromnatural sources, such as naturally nitrate containing plants, e.g. greenleafy vegetables, examples include, but are not limited to spinach,lettuce, fennel, cabbage, Chinese cabbage and beetroot. Concentrates,such as juices or dried concentrates of these and other nitrate-richvegetables or fruits are suitably used for the manufacture of foodproducts (including functional food products) or nutritional supplementsaccording to the present invention.

Polyphenols are a group of chemical substances found in plants,characterized by the presence of more than one phenol group permolecule. Polyphenols are generally further subdivided into hydrolyzabletannins, which are gallic acid esters of glucose and other sugars; andphenylpropanoids, such as lignins, flavonoids, and condensed tannins. Inone embodiment of the present invention the inorganic nitrite and/ornitrate is/are mixed with a compound that contains high levels ofpolyphenols. It is contemplated that this combination will havesynergistic health promoting effects via potentiation of NObioavailability. Polyphenols will enhance NO generation by severalseparate mechanisms highlighted in FIG. 1. First, such agents candirectly stimulate endogenous NO formation from NO synthase enzymes (1in FIG. 1). Second, it is contemplated that these compounds will enhancethe reduction of nitrite to bioactive NO due to the presence ofreductive —OH groups on the phenol ring (2 in FIG. 1). Third, by actingas scavengers of free radicals such as superoxide, they prevent theseradicals from interacting with (and destroying) NO and thereby, NObecomes more long-lived (3 in FIG. 1). In addition to this, nitrite orits reaction products can interact with the polyphenol itself and modifyit chemically via nitration or nitrosation reactions (4 a in FIG. 1).The resulting compound can act as a long-lived NO donor (4 b in FIG. 1).An additional effect is that the presence of polyphenols will divert thechemical reactions away from formation of potentially carcinogenicnitrosamines (5 in FIG. 1). Nitrates reaction product nitrite can reactwith amines to form nitrosamines but polyphenols will inhibit thisreaction by a dual mechanism. First they help to rapidly reduce HNO₂directly to NO thereby minimizing the formation of nitrosating species(N₂O₃, HNO₂). Second, they can directly compete for nitrosation with theamines by being nitrosated themselves.

In one embodiment of the present invention inorganic nitrite and/ornitrate is administered or used in combination with a polyphenol richcompound or product. The ratio nitrite/nitrate comprisingcomposition:polyphenol-rich compound should be chosen to obtain enoughsupply of nitrate. The nitrite/nitrate comprising composition shouldtherefore be at least about 10%, preferably at least about 20%, morepreferably at least about 30%, even more preferably at least about 40%and most preferably at least about 50% or even more. It is contemplatedthat the combination of nitrite/nitrate and polyphenol rich compound orproduct will act synergistically to enhance NO formation in the body atthe expense of detrimental compounds such as nitrosamines. Thebeneficial effects of this includes a reduction in blood pressure andplatelet aggregation, reduced atherosclerosis, reduced risk ofmyocardial infarction, stroke and other cardiovascular disorders,reduced risk of cancer in any form. Examples of polyphenol rich fruit orjuices thereof include, but are not limited to, apple, pear, grapes,lemon, orange, lime, peach, pomegranate, grapefruit, kiwi, ginger andpineapple. Juice from berries are also usable including blackberries,black raspberries, blueberries, cranberries, red raspberries, cherries,bog wortleberry, lingonberries, black elderberry, black chokeberry,black currant, blueberry, cloudberries and strawberries. Other naturalsources of polyphenols include vegetables such as carrots, chili,rhubarb, onions. In addition, cacao products (rich in flavanols), greenor black tea, nuts, Yerba mate and coffee are all rich in polyphenols.In one preferred embodiment the nitrate in the inventive compositionoriginates from beetroot (such as beetroot juice) which is blended withone or several polyphenol-rich compounds or products. The ratio beetrootjuice:polyphenol-rich compound should be chosen to obtain enough supplyof nitrate and therefore the beetroot juice part should be at leastabout 10%, preferably at least about 20%, more preferably at least about30%, even more preferably at least about 40% and most preferably atleast about 50%.

According to one embodiment of the present invention the dose of nitriteand/or nitrate or nitrite and/or nitrate together with polyphenols isadministered or manufactured as a chewing gum, lozenge or pastille,wafer, cake, bar or the like which optionally also comprises livenon-pathogenic bacteria. It can also be administered or manufactured asa functional food product or as a part of a functional food product.

The use of nitrite and/or nitrate or a combination of nitrate and/ornitrite with polyphenols may also have beneficial effects on the bloodpressure. Thus, in one embodiment the present invention relates to amethod to reduce blood pressure, preferably to normal levels (about140/80 mmHg). Examples of nitrite and/or nitrate sources are naturalsources of nitrate (such as vegetables as mentioned above or juicesthereof) or salts of nitrite and/or nitrate. In one particularembodiment beetroot or a juice thereof is administered in order toreduce the blood pressure in a mammal.

The purpose with the combination with non-pathogenic live bacteria is tofurther enhance the generation of bioactive compounds such as NO,nitroso adducts or chemically related compounds. This enhancement willoccur locally in the GI tract via bacteria-dependent reduction ofnitrate and nitrite to NO and other bioactive nitrogen oxides. Inparticular, this combination will be effective in treating andpreventing GI disorders such as ulcers in the stomach, duodenum,jejunum, caecum and colon/rectum. Also, it is contemplated that thiscombined product will be effective in treating and preventinginflammatory bowel disorders including ulcerative colitis, Crohn'sdisease, microscopic colitis and other forms. Irritable bowel disease(IBS) is another condition that could be treated with said product. Inaddition, the compounds formed can be absorbed systemically and havesustained biological effects for example in reducing blood pressure andin preventing atherosclerosis, cancer or any other effect related toenhance NO release as discussed above. Suitable bacteria are the socalled probiotic bacteria, included but not limited to Lactobacilli (forexample L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L.casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L.fermentum, L. reuteri) and Bifidobacteria species such as B. breve, B.bifidum, and B. lactis and probiotic yeasts such a Saccharomycesboulardii. Other suitable non-pathogenic bacteria that enhance nitratereduction or nitrite reduction include e.g. Veillonella species,Staphylococcus species, Actinomyces species or Rothia species. Thesemicroorganisms may also be included in “dry form” for example intablets, capsules, bars, and alike.

In one embodiment a low concentration of ethanol is added to theinorganic nitrite and/or nitrate composition. In one embodiment ethanolis used in combination with or administered together with the inorganicnitrite and/or nitrate composition. It has surprisingly been found thatethanol even in very low concentrations can generate the potentvasodilator ethyl nitrite following reaction with physiological amountsof nitrite. The reaction is enhanced at acidic conditions such as in thegastric lumen. It is contemplated that ingestion of nitrate will lead toaccumulation of nitrite in the saliva and the nitrite will react withethanol in the stomach thereby forming ethyl nitrite. For example, ifthe inventive composition is in the form of a liquid the ethanol contentshould be below about 5% (v/v), more preferably below about 2% (v/v),and most preferable between about 0.5-1.5% (v/v).

In one embodiment of the present invention a cacao product such as darkchocolate that is rich in flavanols is combined with a nitrate-richnatural compound in a drink or a chocolate bar. One preferrednitrate-rich compound in this embodiment is rhubarb. Again, the nitratewill potentiate the effect of the flavanols via enhancement of NOformation as described above and in FIG. 1.

Contamination of a nitrate-containing food or drink with unwantedbacteria may result in a large accumulation of nitrite, due to nitratereducing bacterial enzymes. Ingestion of high levels of nitrite maycause potentially serious methemoglobinemia. In one embodiment anitrate-rich composition is mixed with a compound that inhibits unwantedbacterial growth. Such compound should be chosen so as not to affect thetaste of the product negatively. Ideally, it should enhance the tasteand at the same time increase the bioactivity of the product. One optionis to acidify the inventive composition so that final pH is below about5, and most preferably between about pH 2-4. This will inhibit and/orabolish bacterial growth. Suitable acidifying agents can be any agentthat reduces pH and include artificial compounds as well as naturaljuices from e.g, but not limited to, lemon or lime, ascorbic acid,acetic acid or vinegar (from apple, grapes or other fruits). It iscontemplated that with the use of natural products a dual effect isachieved. Besides having an antibacterial effect, they are rich inpolyphenols, which enhance the generation of bioactive NO fromnitrate/nitrite in the vegetable drink. In one particular embodiment anitrate-rich vegetable juice (e.g. beetroot juice) is mixed with acompound that inhibits undesirable bacterial growth.

According to an embodiment of the invention, the nitrate and nitritesalts are combined with other pharmaceuticals including but not limitedto: anti-diabetic drugs (insulin and oral anti-diabetics),cardiovascular drugs (statins, ACE inhibitors, beta-receptorantagonists, diuretics, angiotensin 2 receptor antagonists, organicnitrates, calcium channel blockers) acid secretion-inhibitors (protonpump inhibitors, Histamine-2 receptor blockers), oral anti-diabeticsincluding biguanides, sulphonureides, alpha-glucosidase inhibitors,thiazolidinediones, glinides; drugs for treatment of pulmonaryhypertension including prostacyclin analogues, endothelin receptorantagonists and sildenafil.

According to one embodiment the nitrite is delivered systemically viaperoral treatment with an organic nitrate including nitroglycerine orisosorbide mono/di-nitrate. Nitroglycerine is used clinically to treatangina pectoris and it acts by releasing vasodilatory nitric oxidesystemically. However, this drug must be given parenterally because thefirst passage metabolism in the liver is considerable. Interestingly, ithas been found that the liver metabolism of nitroglycerine yieldspredominantly nitrite. Considering the novel biological effects ofnitrite described here, nitroglycerine may therefore be used as a“prodrug” of nitrite. Preferably the drug should then be given by theenteral route (with liver metabolism) to maximize nitrite generationwhile at the same time avoiding the acute vasodilatation and drop insystemic blood pressure associated with iv or sublingual administrationof nitroglycerine. A suitable dose range when giving nitroglycerineperorally is about 0.001 to 10 mol/kg/24 hours, preferably about 0.001to 1 mol/kg/24 hours, more preferably 0.01 to about 0.1 mmol/kg/24hours. The tablets should preferably be coated to avoid absorption inthe oral cavity.

According to one embodiment nitrite and/or nitrate is added toparenteral and enteral feeding/nutrition solutions to be used in adults,children, neonates and prematures. Today such solutions are generallyextremely low in nitrate and nitrite as noted in measurements performedby the present inventors (not shown). An intubated mechanicallyventilated patient with parenteral nutrition is particularly deprived ofnitrate/nitrite. First, these patients do not produce and swallow salivaproperly and thus one great nitrate/nitrite source is disrupted.Secondly as stated above, the feeding they receive contains almost nonitrate/nitrite. Many intensive care patients suffer from metabolicdisturbances, in particular catabolism due to stress and trauma andtheir metabolic rate is often increased. Moreover, insulin resistance iscommon and glucose homeostasis is disturbed. Tight control of plasmaglucose by administering insulin is advocated in these patients. Alsohealthy subjects, such as infants or subjects taking enteral solutionsfor other reasons can benefit from the compositions and methods of thepresent invention. Enteral nutrition is thus meant to include alsoinfant formulas and other enteral products.

In one embodiment, provided herein are uses of nitrites and/or nitrates,for the manufacture of pharmaceutical products, enteral or parenteralnutritional solutions, preoperative compositions, nutritionalsupplements, or functional food products for administration to bothhealthy persons, such as athletes, or to patients, suffering from one ormore of the conditions exemplified herein. Included in this embodimentare also possible combinations of nitrites and/or nitrates with othercompounds as mentioned above.

In one embodiment, provided herein are methods for the treatment,alleviation and/or prevention of clinical conditions, comprisingadministering an effective amount of a nitrate and/or a nitrite to apatient in need thereof sufficient to treat, alleviate and/or preventsuch condition.

There are many possible clinical conditions where such treatment,alleviation and/or prevention is performed using nitrate and/or nitriteaccording to the present invention:

-   -   glucose control in diabetes/prediabetes    -   metabolic syndrome

The method and composition according to the invention also has generaltherapeutic, alleviating and/or prophylactic effects in patients undermetabolic stress. Examples include but are not limited to:

-   -   intensive care patients    -   patients undergoing surgery    -   patients suffering from malnutrition of different genesis    -   cancer with anorexia    -   burn injury    -   trauma    -   neonates/prematures    -   anorexia nervosa    -   thyreoidea disorders (e.g. hyperthyreosis)    -   myocardial infarction, cardiac arrest    -   major systemic disease with a catabolic state    -   stress ulcers (gastric)    -   surgical gut anastomosis insufficiency    -   fever    -   myocardial infarction and cardiac arrest    -   ischemia-reperfusion injury (MI, stroke, arterial insufficiency        or any other organ ischemia)    -   sleep apnoea syndrome    -   septic chock    -   insufficient perfusion of the intestines

Patients treated in the ICU (intensive care unit) are often subjected tosevere metabolic stress due to trauma, infection, pain and otherpathological processes. Such patients are treated for several reasonsincluding but not limited to post surgical observation, trauma,bleeding, burn injury, brain injury, stroke, diabetes, sepsis, septicshock, myocardial infarction, cardiac arrest, arterial insufficiency orany other organ ischemia, chronic obstructive pulmonary disease, asthmaand other severe inflammatory conditions, pulmonary hypertension,congestive heart failure, pulmonary embolism. This results in varyingdegrees of catabolism and resistance to insulin with a disturbed glucosehandling. They also often suffer from vascular endothelial dysfunctionleading to microcirculatory disturbances. As mentioned above thesepatients are given only minute amounts of nitrate/nitrite in the enteraland parenteral feeding and due to several reasons (sedation, intubation)they have a disturbed enterosalivary circulation of nitrate/nitritecompared to healthy people.

It is contemplated that addition of sufficient amounts of nitrite and/ornitrate in the parenteral and/or enteral feeding to patients treated inthe intensive care unit, may alleviate or prevent the aforementionedmetabolic and circulatory disturbances by decreasing the rate ofmetabolism, enhancing blood glucose homeostasis and microcirculatoryimprovement.

Patients undergoing surgery are subjected to a varying degree ofsurgical trauma. This triggers catabolic hormones like cortisol,glucagon and adrenalin and such patients may develop transient insulinresistance. Common clinical procedure involves fasting in order toreduce gastric content which could be accidentally aspirated in theairways if the patient vomits or regurgitates during anaesthesia. Suchfasting eliminates the intake of nitrate/nitrite and it has been shownthat systemic levels of nitrate and nitrite are reduced after fasting.It is contemplated that administration of nitrite and/or nitrate pre,per or postoperatively will improve the metabolic situation (decreasingthe rate of metabolism, enhancing blood glucose homeostasis) in surgicalpatients. Moreover, during the entire perioperative period the patientsare at higher risk for ischemic events due to hypotension, hypoxia andmicrocirculatory disturbances. Such events will initiateischemia-reperfusion processes which may injure several organ systemswithin the body. It is contemplated that prophylactic administration ofnitrite and/or nitrate, either as a preoperative drink containingnitrate and/or nitrite anions, a suitable vegetable juice such as butnot limited to beetroot juice or by giving nitrate and/or nitrite viathe parenteral route, will reduce the negative effects ofischemia-reperfusion events during surgery in several organs includingbut not limited to brain, heart, lung, liver, kidney and skeletalmuscle.

Ischemia-reperfusion injury during myocardial infarction is a majorproblem in clinical care. State-of-the art treatment of myocardialinfarction includes reopening of occluded coronary vessels bypharmacological means with thrombolytic agents and/or percutaneouscoronary intervention. It is considered that the method and compositionaccording to the invention will reduce ischemia-reperfusion injury byincreasing blood flow and by altering mitochondrial function. Fromexperiments in the present application it is also considered that duringischemia-reperfusion nitrate and nitrite could reduce oxygen consumptionpossibly by a “hibernating” effect on mitochondria. Another mechanism bywhich nitrate and nitrite could ameliorate ischemia-reperfusion injuryis by reducing oxygen radical formation and cytochrome C release fromthe mitochondria. The effect of nitrate and nitrite on mitochondrialfunction is most likely mediated by nitric oxide (NO) interacting withoxidative phosphorylation and/or s-nitrosylation of protein complexes inthe mitochondrial respiratory chain. It is envisaged that the time pointfor administration of nitrate and/or nitrite in relation to themyocardial infarction induced ischemia-reperfusion injury will beeffective both pre, per or post injury. Both a pre and post conditioningeffect of nitrate and/or nitrite is considered which means that apatient can receive treatment already in the ambulance on his/her way tothe hospital, at the hospital before and after reperfusion of thecoronary circulation and also at the ward. Also patients at high risk ofdeveloping a myocardial infarction (eg. angina pectoris, congestiveheart failure) could be considered for prophylactic treatment with themethod and composition according to the invention. After cardiac arrestand resuscitation systemic ischemia-reperfusion injury developsinvolving several major organ systems, including the brain. It iscontemplated that nitrate and/or nitrite is beneficial both givenprophylactic to patients with high risk for cardiac arrest and alsoduring resuscitation procedures.

According to a preferred embodiment, the nitrate and/or nitrite or anycombination mentioned herein is/are also given pre-operatively topatients scheduled to undergo surgery or substantive, invasiveexamination procedures. In one particular embodiment, inorganic nitriteand/or nitrate are combined with carbohydrates. Thus, such combinationsinclude, but are not limited to nitrate alone; nitrate and nitrite;nitrate, nitrite, and carbohydrates; nitrate and carbohydrates; nitritealone and nitrite and carbohydrates. In addition polyphenols may beadded to any of the aforementioned combinations. Any such combinationcan be administered (in single or repeated doses) as a preoperativedrink or the like prior to surgery or intravenously prior or duringsurgery. An appropriate time to administer such a preoperative drink isbetween 72 and 2 hours before surgery. A combination of nitrite and/ornitrate with carbohydrates may ameliorate insulin resistance and alsoischemia/reperfusion injury common in the preoperative and postoperativeperiod. Examples of carbohydrates include but are not limited toglucose, fructose, maltodextrin, sucrose, lactose, galactose andmannose. When given preoperatively the amount of carbohydrates in thedrink will provide the patient with preferably about 200 kcal, morepreferably about 100 kcal and most preferably about 50 kcal. For the useof a nitrate salt perorally, a dose of about 0.01-100 mmol/kg/24 h iscurrently preferred or more preferably a dose of about 0.01-10mmol/kg/24 h, even more preferably 0.1-1 mmol/kg/24 h.

Using intravenous administration pre and/or peroperatively, the nitriteis preferably administered in a dose within the interval of about 0.01to about 10 000 nmoles/kg/min, preferably about 0.01 to about 1000nmoles/kg/min, more preferably about 0.1 to about 100 nmoles/kg/min,most preferably about 1 to about 20 nmoles/kg/min. It is presentlycontemplated that the most preferred dose is less than about 15nmoles/kg/min

In patients suffering from arterial insufficiency, including but notlimited to intermittent claudication, pharmacological treatment aims atimproving blood flow in the affected limb(s) and to stimulateangiogenesis to promote new vessel formation. Phosphodiesteraseinhibitors and growth factors have been studied in clinical trials butresults are variable. Likewise, studies with traditional NO donors suchas organic nitrates have been less successful. It is contemplated thatthe method and composition according to the invention will positivelystimulate both blood flow and angiogenesis thereby improving thecondition in these patients. Treatment with nitrate/nitrite isattractive since it will preferentially increase blood flow in theischemic areas without causing troublesome systemic effects such ashypotension which is a risk when using organic nitrates that dilatenon-selectively in most vascular beds. In addition, nitrite will affectmitochondrial function as discussed above resulting in less oxygendemand in the ischemic tissue.

In patients suffering from malnutrition, including but no limited tocancer with anorexia, anorexia nervosa, gastrointestinal disease, it iscontemplated that the method and composition according to the inventionwill have an anabolic effect and will reduce oxygen consumption. It iscontemplated that this effect is achieved by improving mitochondrialefficiency and attenuating mitochondrial uncoupling.

The method and composition according to the invention also has generaltherapeutic effects in patients with erectile dysfunction. In thiscondition the endogenous NO system is failing. Administration of nitriteand/or nitrate (and possible combinations outlined above) will enhanceNO formation locally in the corpus cavernosum, thereby enhancingerection.

It is contemplated that the method and composition according to theinvention has general therapeutic, alleviating and/or prophylacticeffects in patients with gastritis and gastric ulcers due toHelicobacter pylori infection, stress or side-effects frompharmacological treatment such as the use of Non-SteroidalAnti-Inflammatory Drugs (NSAID). By improving gastric mucosal bloodflow, mucus generation and by anti-bacterial and antiinflammatoryproperties the method and composition will have beneficial effects inthese patients. In addition unwanted side-effects of treatment withacetylsalicylic acid, NSAIDs or any other ulcerogenic drug in otherparts of the gastrointestinal tract (duodenum, small and largeintestines) are prevented by said method and composition.

A feared problem in surgery is gut anastomosis insufficiency andinsufficient perfusion of the intestines. New methods for improvingmicrocirculation in the anastomosis are constantly investigated. It iscontemplated that the method and composition according to the inventionwill improve microcirculation in the anastomosis which will enhance thehealing process. It is also considered that the invention will improvecirculation in situations with insufficient perfusion of the intestines.

Patients with sleep apnoea syndrome are at higher risk for developinghypertension and other cardiovascular diseases. Moreover, they aresubjected to periods of hypoxia during sleep. It is contemplated thatthe method and composition according to the invention will protectagainst hypoxia-induced injury by improving circulation and by reducingoxygen consumption possibly by a “hibernating” effect on mitochondria.Other mechanism by which said method and composition could haveprotective effects is by reducing oxygen radical formation andcytochrome C release from the mitochondria. The effect of nitrate andnitrite on mitochondrial function is most likely mediated by nitricoxide (NO) interacting with oxidative phosphorylation and/ors-nitrosylation of complexes in the mitochondrial respiratory chain.

The method and composition according to the invention has generaltherapeutic, alleviating and/or prophylactic effects also in patientswith pathological conditions characterized by low oxygen availability,including but not limited to chronic obstructive airway disease (COPD),inflammatory airway disease such as asthma, pulmonary hypertension,congestive heart disease, interstitial lung disease, pulmonary embolism,ischemic heart disease, peripheral artery disease, sleep apnéa syndrome,mycocardial infarction and systemic inflammatory disorders. In theseconditions addition of oxygen promptly improve arterial oxygenation andtotal body oxygen delivery. For technical and safety reasons it iscomplicated to administer oxygen outside hospital facilities. Anotherway to improve the situation for these patients is to reduce the needfor oxygen. The method and composition according to the invention leadto reduced oxygen cost in relation to the physical work performed. Thishighly surprising finding is especially relevant in the patients withthe aforementioned conditions since oxygen availability is the limitingfactor for physical activity. It is envisaged that the method andcomposition according to the invention will facilitate physical activityin these patients groups.

In one preferred embodiment nitrate and/or nitrite is given to patientswith a pathological condition characterized by low oxygen availability.In such situations it is desirable to reduce the tissues need for oxygento prevent sequele and symptoms associated with the hypoxia. Examplesinclude patients with COPD whose pulmonary oxygen uptake may be severelycompromised and patients with peripheral artery disease where oxygendelivery to the tissues is reduced.

The inventive method and composition is also useful for healthysubjects, e.g. athletes. The inventive method and composition providesfor less oxygen demand at a certain workload and improves anabolism. Themethod and composition is also useful for oxygen sparing at highaltitude, for example in work and sports performed in a low oxygenenvironment, such as but not limited to rescue activities, firefighting, military operations, diving, mountain climbing, high-altitudeflying, and the exploration of space.

The present method and composition is also useful in solid organ ortissue transplantation, in order to minimize metabolic demand of thedonated organ before transplantation, and to improve survival of thetransplanted organ or tissue after transplantation. Nitrite and/ornitrate or any combination mentioned herein is/are given either into theorgan or tissue by perfusion, topically on the organ or tissue and/orsystemically to the donor before transplantation, and into the organ ortissue by perfusion or topically on the organ or tissue and/orsystemically to the receiver after transplantation.

In one preferred embodiment nitrate and/or nitrite is/are or anycombination mentioned herein given to patients that are at risk ofdeveloping significant metabolic stress. Such situations include many ofthe conditions listed above including surgical stress and trauma. Oxygendemand and consumption increases dramatically during stress. Thus, animproved situation is achieved by decreasing the oxygen demand in thesepatients. According to one preferred embodiment nitrate and/or nitriteis given to patients to prevent the sequele associated with physiologicor traumatic stress. Examples include patients that come into theemergency room after trauma or patients undergoing major surgery.

To the best of the knowledge of the inventors, this is the first studyto examine the effects of dietary nitrate on the cardiopulmonary andmetabolic response to exercise. The main finding was that dietarysupplementation with inorganic nitrate, in an amount which does notcause significant hypotension and without any significant increase inmethemoglobin and plasma lactate, results in a reduced VO₂ duringsubmaximal work and a significant increase in muscular efficiency. Theseeffects occurred without any changes in VO_(2peak) values or maximalattainable work rate.

Without wishing to be bound by theory, the inventors consider that thereis reason to believe that the observed effects involve initial reductionof nitrate to nitrite. Nitrate itself is believed to be biologicallyinert and cannot be metabolised by mammalian cells. However, afteringestion nitrate re-enters into the mouth via the salivary glands andis effectively reduced by commensal bacteria thereby forming nitrite. Incontrast to nitrate the nitrite ion has recently been shown to possess awide range of bioactivities. In the present study the inventors didindeed note an increase in plasma nitrite after the nitrate treatmentperiod thereby confirming in vivo reduction of nitrate as describedpreviously. Another finding in support of nitrite being bioactive wasits effective consumption during exercise in contrast to the unchangedlevels of plasma nitrate. Ultimately the bioactivity of nitrite islikely related to its further reduction to NO and possibly other closelyrelated nitrogen intermediates. In addition, it has been recentlysuggested that, nitrite itself may directly affect cellular signalingpathways. Although probably unlikely, at this stage effects of thenitrate ion itself cannot be excluded. There are several principle waysby which biological effects of nitrogen oxides may be propagatedincluding activation of cGMP, alteration of protein function by anitro(syl)ation/nitration or direct binding to protein heme-moieties ofseveral proteins as in the prototypic activation of guanylyl cyclase byNO. In addition, nitrite itself may also directly affect cellularsignaling pathways.

If the effects proceed via nitrate reduction to nitrite and then NOformation, how could this then explain the present results? Earlierstudies using NOS inhibitors to block endogenous NO production give someindications. NOS-inhibition has been shown to increase submaximal VO₂ indogs during exercise, independently of the reduction in blood flow. Thisincrease in VO₂ during NOS-blockade has been linked to the fact that NOaffects tissue respiration in vitro by reversible inhibition of therespiratory enzyme cytochrome c oxidase. Others have related theincreased VO₂ during NOS-blockade to an inhibiting effect of NO onproton leakage via the mitochondrial permeability transition pore (mPTP)were a considerable amount of protons leak over the inner mitochondrialmembrane. If the effects of nitrate were solely due to inhibition ofcytochrome c oxidase (thereby inhibiting oxidative phosphorylation) onewould expect an increase in anaerobic metabolism during physicalexercise and a larger accumulation of lactate. However, judging from theresults this was not the case, as the plasma lactate concentration wasnear identical after nitrate supplementation compared to placebo.

The finding that the oxygen pulse at a given work rate decreases bynitrate supplementation is a direct effect of the lower oxygen demand atthat work rate. However, there is no difference in oxygen pulse at agiven absolute oxygen uptake. The lack of effect of nitrate on VE/VO₂ oroxygen pulse indicates that the improved efficiency originates frommuscular or mitochondrial adaptations rather than from centraladaptations in the heart or the lungs.

EXAMPLES

The present inventors have been studying the effects of nitrate andnitrite on various physiological functions including blood pressure,glucose metabolism and energy expenditure in vivo. In the examples belownitrate was administered perorally and nitrite was administeredintravenously (iv). Oxygen consumption was measured using indirectcalorimetry. The term “indirect calorimetry” is here defined as a methodfor calculating heat that living organisms produce from their productionof carbon dioxide and nitrogen waste and from their consumption ofoxygen, well known to persons skilled in the relevant art.

1. Intravenous Sodium Nitrite and Resting Energy Expenditure

Resting energy expenditure (as measured by indirect calorimetry) isreduced by 10-25% after 10 min of iv infusion of sodium nitrite (n=4,FIG. 3). Prior to the test the subjects had been on a diet low innitrate for one day and had been fasting for at least 8 h. The majordrop in energy expenditure was seen when infusing nitrite at aconcentration of 10 nmoles/kg/min during 10 minutes. At 1 nmol/kg/min noobvious effects were noted during the 10 min observation period andafter 100 nmol/kg/min no further decrease in oxygen consumption wasnoted as compared to the 10 nmol dose. In separate experiments (n=2) thepresent inventors noted an increase in plasma nitrite from 140-165 nM to370-480 nM after the 10 nmol/kg/min infusion (10 min infusion). Basallevels of methemoglobin were 1.1-1.3 mmol/l and did not changesignificantly after infusion of nitrite (1.1-1.4 mmol/l) See FIG. 2.

2. Oral Sodium Nitrate and Oxygen Consumption During Exercise

Methods

Subjects: Nine healthy, well-trained (VO_(2peak) 55+/−3.7ml×kg⁻¹×min⁻¹), males (28+/−6 years) volunteered for the study. Allsubjects were trained cyclists or triathletes and well accustomed to thetesting procedure. It was chosen to use well-trained subjects to avoidtraining effects from the tests such as enhanced VO_(2peak) or bettermechanical efficiency during submaximal exercise. The protocol wasapproved by the regional ethics committee in Stockholm and all subjectsgave their written consent prior to participation.

Dietary supplementation with nitrate: The aim with the present study wasto investigate the effects of two distinct dietary patterns, one withhigher, and one with lower than normal nitrate intake. The study had adouble-blind placebo-controlled cross-over design. During two three-dayperiods, separated by a washout interval of ten days, the subjects wereinstructed to avoid all foods with moderate or high nitrate content (allvegetables, all cured meats, strawberries, grapes, and tea). Inaddition, they were told to restrain from alcohol and tobacco products.Otherwise they were free to eat any food they liked during the threedays of restricted diet. The subjects were randomized to start witheither ingestion of 0.1 mmol sodium nitrate/kg bodyweight/day dissolvedin water or an equal amount of sodium chloride (placebo). The daily dosewas divided and ingested three times daily. The different solutionscould not be distinguished by taste or appearance. The daily nitratedose corresponded to the amount normally found in 150-250 gram of anitrate-rich vegetable such as spinach, lettuce or beetroot. The lastdose of nitrate or placebo was ingested in the morning on the day ofmeasurement (see the main tests below). The order between the nitratesupplementation period (NIT) and the placebo period (CON) was balanced.During the washout period the subjects did not adhere to any specificdietary regime.

Experimental protocol: Measurements were carried out on an electricallybraked cycle ergometer (Monark 839E, Varberg, Sweden) that was modifiedwith a racing saddle and the pedal system the subjects were familiarwith from training. The bicycle ergometer was computer-controlled,permitting a constant work rate regardless of the cadence the subjectchose to pedal with. The pedaling cadence was individually chosen in therange of 70-90 rpm but kept constant during the test to minimizedifferences in work output due to changes in muscle recruitmentpatterns.

Pulmonary ventilation (V_(E)), oxygen uptake (VO₂), CO₂ output (VCO₂)and respiratory exchange ratio (RER) were measured at 10 secondintervals by a computerised gas analyser (AMIS 2001, Odense, Denmark)connected to a flow meter which the subjects breathed through via amouthpiece and a plastic tube. Heart rate (HR) was continuously recordedduring the tests with a portable heart rate monitor (Polar S610, Polar,Kempele, Finland). Capillary blood samples (20 μl) were collected fromthe fingertip and were analyzed for lactate ([HIa]) using a BiosenC-Line Sport Analyser (EKF diagnostics, Magdeburg, Germany). Haemoglobinconcentration ([Hb]) at rest was determined with capillary blood takenfrom the fingertip and analyzed with an Hb-measuring device (Hemocue,Ängelholm, Sweden). Hematocrit (Hct) was determined by centrifugingcapillary blood at 12000 rpm for three minutes.

Pre-tests: Each subject attended the laboratory twice within a two weekperiod before the first main tests. The first pre-test was carried outto familiarize the subject with the bicycle ergometer and the testingprocedure. The subjects did a preliminary test at five submaximal levelswith every level lasting for five minutes. There was no rest between thedifferent submaximal levels. VO₂ was continuously measured with the AMIS2001. At the end of each submaximal level capillary blood was taken fromthe fingertip and later analysed for [HIa]. At every work rate thesubjects rated their perceived exertion on the Borg's RPE-scale (BORG,G. Perceived exertion as an indicator of somatic stress. Scand J RehabilMed. 1970, vol. 2, no. 2, p. 92-8), both central and muscular exertionwere rated. After eight minutes of recovery, the subject was instructedto cycle for as long as possible at a work rate corresponding to hiscalculated maximal oxygen uptake (ÅSTRAND, P-O, et al. Textbook in workphysiology. McGraw-Hill, 1970. ISBN 0070024065. p. 619). During thistest the subjects actual VO_(2peak) was measured and if the subject wasable to cycle for longer than seven minutes extra power of 20-30 wattswas added every minute until exhaustion. One and three minutes after themaximal test capillary blood were sampled from the fingertip foranalysis of [HIa].

Before the second pre-test, the submaximal levels were adjusted so thatthey corresponded to 45, 60, 70, 80 and 85% of VO_(2peak). The maximalwork rate was also adjusted, if necessary, so that the time toexhaustion was kept between four and seven minutes.

The main tests: The subjects refrained from heavy exercise three daysprior to the main tests and avoided all exercise the day before thetests. They were also told to eat their last light meal at least 3 hoursbefore the start of the tests. When the subjects came to the laboratorythey received their last dose of either placebo or nitrate and wereallowed to rest in the supine position for 60 minutes before the testcommenced.

All subjects used a standardised warm up procedure of five min ofcycling at 100 watts followed by five minutes of rest. The submaximaland maximal tests were performed in the same way as the second pre-testwith five submaximal work rates lasting five minutes each, without restbetween the different levels. Identical work rates were used during thetwo main tests. Venous blood (9 ml) was drawn at rest 45 minutes afterthe last nitrate/placebo-dose was ingested and again immediately afterthe VO_(2peak)-test. The blood was placed in an ice bath and centrifugedwithin five minutes at 1300 rpm and 4° C. The plasma was separated andkept at −80° C. until it was analysed for its nitrate and nitriteconcentrations by a chemiluminescence assay as described previously(LUNDBERG, J O, et al. Inorganic nitrate is a possible source forsystemic generation of nitric oxide. Free Rad Bio Med. 2004, vol. 37,no. 3, p. 395-400).

Statistics and calculations: Results are expressed as means+/−standarddeviation (mean+/−SD). Paired t-tests were used to evaluate thedifference between the nitrate and the placebo trials. The significancelevel was set as p=<0.05.

Gross efficiency (GE) was defined as the work rate divided by the actualenergy expenditure (EE). The EE was in turn calculated with the Brouwerequation (BROUWER, E. On simple formulae for calculating the heatexpenditure and the quantities of carbohydrate and fat oxidized inmetabolism of men and animals, from gaseous exchange (Oxygen intake andcarbonic acid output) and urine-N. Acta Physiol Pharmacol Neerl. 1957,no. 6, p. 795-802). Delta efficiency (DE) was defined as the increase inwork rate divided by the increase in EE (GAESSER, G A, et al. Muscularefficiency during steady-rate exercise: effect of speed and work rate. JAppl Physiol. 1975, no. 38, p. 1132-1139). The DE was based on the fourlowest work rates and was analyzed with linear regression. The oxygenpulse is defined as VO₂/HR.

Results

Blood pressure at rest: Average resting systolic blood pressure waslower after nitrate supplementation (112+/−8 mmHg) compared to placebo(120+/−5.9, p<0.01). The diastolic blood pressure was also lower afternitrate (68+/−5.5 mmHg) compared to placebo (74+/−6.8 mmHg, p<0.01).Parts of these findings have been published as a separate communication(Larsen et al. 2006).

Blood values: No change was observed in [Hb] at rest (NIT 152+/−11, CON153+/−11 g×l⁻¹, p=0.87) or immediately after the VO_(2peak)-test (NIT163+/−13, CON 161+/−13 g×l⁻¹, p=0.27). Nor were there any change in thehematocrit value at rest (NIT 42+/−4, CON 43+/−3%, p=0.19) or after theVO_(2peak)-test (NIT 46+/−4, CON 47+/−4%, p=0.6).

Plasma levels of nitrate at rest were 27+/−6.9 μM in CON and 182+/−55 inNIT (p=<0.01). Nitrate levels immediately after the maximal work testwere 29+/−6.1 in CON and 175+/−61 μM in NIT (p=<0.01). Plasma nitratedid not change during exercise either in NIT or in CON (p=0.8). Nitritelevels at rest were 124+/−28 in CON and 226+/−87 nM in NIT (p=<0.01).Immediately after the maximal work test the nitrite levels were 111+/−29in CON and 137+/−48 in NIT (p=0.17).

The decrease in nitrite concentrations during exercise was morepronounced in NIT than in CON (FIG. 6). The increase in c-GMPconcentrations after the maximal work as compared to rest tended to behigher in NIT than in CON (p=0.08).

Blood pressure: Average resting systolic blood pressure decreased from120+/−5.9 after NIT to 112+/−8 mmHg after CON (p=0.003). The diastolicblood pressure decreased from 74+/−6.8 to 68+/−5.5 mmHg in the CON andNIT-groups respectively (p=0.005). Parts of these findings have beenpublished as a separate communication (LARSEN, F J, et al. Effects ofdietary nitrate on blood pressure in healthy volunteers. N Engl J Med.2006, vol. 355, no. 26, p. 2792-3).

Submaximal work parameters: After nitrate administration VO₂ wassignificantly lower during the four work rates corresponding to 45-80%VO_(2peak) compared to the placebo period (FIG. 5). The most significantdifference was seen at 80% of VO_(2peak) (NIT 3.44+/−0.31 l×min⁻¹ vs CON3.61+/−0.31 l×min⁻¹, p=0.003, FIG. 5). On average VO₂ were was 0.15l×min⁻¹ lower in the NIT-trials over the 4 submaximal work rates. Therewas no difference in heart rate (HR) between the NIT and CON-trials (seeFIG. 6). The oxygen pulse tended to decrease from 21.0+/−2.0 during CONto 20.3+/−1.9 ml×beat⁻¹ (p=0.08). No significant differences changeswere found between NIT and CON in [HIa] (FIG. 6), V_(E), V_(E)/VO2 orrespiratory exchange ratio (RER) during any of the submaximal workrates. The average Gross efficiency (GE) is defined as the work ratedivided by the actual energy expenditure (EE). The EE was in turncalculated with the Brouwer equation (Brouwer, supra). GEgrossefficiency improved from 19.7% during CON to 21.1% during NIT (p=0.02).Delta efficiency (DE) is the increase in workload divided by theincrease in EE (Gaesser & Brooks, supra). The DE is in this case basedon the four lowest work rates which were analyzed with a linearregression analysis. The DE Delta efficiency (DE) increasedsignificantly from 22.1+/−1.6% during CON compared to 22.9+/−1.9% duringNIT, (p=0.04).

Maximal work capacity: There was no significant difference in theVO_(2peak) between the NIT and CON trials (4.49+/−0.44 and 4.61+/−0.28l×min⁻¹ respectively, p=0.29). These values were also not significantlydifferent from the VO_(2peak) achieved during the pre-test (4.54+/−0.32l×min⁻¹). Likewise, no significant differences were noted either inV_(Emax) (NIT 182+/−21.4 vs CON 186+/−21.7 l×min⁻¹, p=0.5), HR_(max)(NIT 189.8+/−7.0 vs CON 190.3+/−7.5 beats×min⁻¹, p=0.94) or maximal workrate (NIT 360.6+/−32.8 vs CON 358.9+/−32.3 watt, p=0.35). There was nodifference between NIT and CON in the rating of perceived exertion (BorgRPE-scale) at any work load (submax or max).

Comment to the results: In the present study a significantly reducedoxygen demand at submaximal workloads was noted after nitrateadministration was noted at the four lowest workloads. The fifth workrate, at approximately 85% VO_(2peak), was well above the lactatethreshold in several subjects and thus the anaerobic energy productionbecame more pronounced. This led to involvement of accessory musclegroups and a noticeably change in motion pattern. At this work rate theVO₂ did not reach a stable steady-state level and is thereforeunsuitable for the calculation of muscular efficiency. The reason forincluding this fifth work rate in the protocol was to receive a lactatevalue above the lactate threshold and thereby get an indication ofchanges in the upper part of the lactate curve.

3. Oral Sodium Nitrate and Glucose Homeostasis

The increase in plasma glucose after a standard Oral Glucose ToleranceTest (75 g glucose in 250 ml water) is lower when measured after nitratepre-treatment (1 mmol/kg NaNO₃) compared to placebo (NaCl). The studywas performed with three healthy test subjects in a double-blind study.Glucose ingestion started 60 min after the nitrate ingestion. Bloodglucose levels were measured during the 120 min period after the glucoseload (n=3). At 30 min mean plasma glucose was 8.2 mmol/l with placeboand in the same subjects 6.5 mmol/l after nitrate supplementation. Theresults are shown in FIGS. 7a, b , and c.

The lower increase in plasma glucose after nitrate pre-treatment wasverified in a further study wherein a standard Oral Glucos ToleranceTest (75 g glucose) was performed in 8 healthy non-smoking subjectsafter a three-day supplementation with either sodium chloride or sodiumnitrate at equal molar amounts (0.1 mmol/kg/day). The study had adouble-blind, placebo-controlled, cross-over design. Blood glucoselevels were measured at two time points before (−10 and 0 min) and at15, 30, 45, 60, 75, 90, 105 and 120 min after glucose intake (n=8). Atthe occasion when the subjects had taken nitrate, the area under thecurve for blood glucose was smaller compared to when they had ingestedplacebo. The results are shown in FIG. 8 as mean±SEM.

4. Effect of Ingestion of Beetroot on Blood Pressure

A 43 year old non-smoking subject with hypertension ingested freshbeetroot juice (300-400 ml/day) for 14 days. The blood pressure wasmeasured twice a day for 14 days and then twice on day 20 (counted fromday 1 of treatment). The Basal blood pressure was 142/99 on the day whenthe experiment started.

The results from the ingestion of beetroot juice are shown in FIG. 9.The mean of the two daily measurements are shown in the bars. Withingestion of the beetroot juice (between day 1-14) a mean reduction insystolic pressure of ≈15 mmHg and a mean reduction in diastolic pressureof ≈16 mmHg was seen. When measured again 6 days after stopping beetrootjuice treatment (day 20), the blood pressure had increased to basallevels (140/100). Pulse rate was unchanged throughout the experimentalperiod. MAP=mean arterial pressure.

5. Concentrations of Plasma Nitrite after Intravenous Infusion ofNitrate

Methods

Anesthetised rats (n=11) were given a bolus dose (10 mg/kg body weight)of NaNO₃ (open boxes) and blood samples were collected at indicated timepoints. 7 additional rats were pre-treated with 30 mg/kg body weight ofallopurinol given intra peritoneally 60 minutes prior to the NaNO₃infusion (closed triangles) and blood samples were collected at the timepoints indicated. Allopurinol inhibits the xanthionoxidase, an enzymesuggested being involved in the reduction of nitrate to nitrite inmammal cells. Plasma was extracted and analysed for nitrate and nitrite.

Three different strains of mice—wild type (n=5 placebo, n=5 nitrate),germ free (n=5 placebo, n=5 nitrate) and eNOS knockout mice (n=2placebo, n=3 nitrate), were given an intra peritoneal injection of 10mg/kg nitrate or placebo (NaCl) and plasma level of nitrite measured 1hour later.

Results

The results from the intravenous infusion of nitrate are shown in FIG.10 a-c. FIG. 10a shows the plasma nitrate concentration and FIG. 10bshows the plasma nitrite concentration. After infusion of nitrate theconcentration of plasma nitrate increases dramatically both in rats thatreceived nitrate and rats that received nitrate+allopurinol (a). Theplasma nitrite concentrations increased in rats that received nitrate aswell as in rats that received nitrate+allopurinol, (b). However theincrease in the rats that received only nitrate was significantlygreater, *p<0.05.

FIG. 10c shows that nitrate-induced increase in plasma nitrite is equalin wild type (n=5 placebo, n=5 nitrate), germ free (n=5 placebo, n=5nitrate) and eNOS knockout mice (n=2 placebo, n=3 nitrate), p=0.05*.

Previously it has been suggested that only bacterial cells and notmammalian cells can reduce nitrate to nitrite. These resultssurprisingly show that also mammalian cells can metabolise nitrate tonitrite. Further, they suggest that the xanthionoxidase enzyme isinvolved in the reduction of nitrate to nitrite.

6. Enhancement of Post-Ischemic Blood Flow

Rats received an intravenous bolus dose of 10 mg/kg nitrate (NaNO₃, n=4)or placebo (NaCl, n=4)) diluted in PBS (pH 7.4) followed by continuousinfusion of 3 mg/kg/h. 60 minutes after the addition of nitrate (openboxes) or placebo (filled circles), L-NAME (50 mg/kg) was given and 10minutes later a supra renal clamping of the abdominal aorta wasperformed. After 30 minutes of ischemia the clamp was removed and theabdominal aortic blood flow was monitored during 60 minutes.

The results show that the nitrate-treated rats maintain a higher bloodflow during the early (0-10 minutes) as well as the late (10-60 minutes)post-ischemic phase compared to the placebo treated rats (FIG. 11).Remarkably, after 60 min of reperfusion the blood flow had decreased toonly 20% of pre-ischemic values in the control rats, while in thenitrate-treated rats, the blood flow was maintained at almost 75% ofcontrol values. This demonstrates a strong augmentation of thenitrate-nitrite-NO pathway during an ischemic event.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionas set forth in the claims appended hereto.

REFERENCES

-   WO 2005/004884 A (US GOVERNMENT ET AL.) 20 Jan. 2005-   WO 2005/007173 A (US GOVERNMENT ET AL.) 27 Jan. 2005-   Joint FAO/WHOExpert Committee on Food Additives (JECFA). Safety    Evaluation of Certain Food Additives. WHO, 1970. ISBN 9241660503.-   TANNENBAUM, S. R., et al. Nitrite in human saliva. Its possible    relationship to nitrosamine formation. J cancer Ins. 1974, vol.    53, p. 79-84.-   BARTSCH, H., et al. Inhibitors of endogenous nitrosation: mechanisms    and implications in human cancer prevention. Mutation Res. 1988,    vol. 202, p. 307-324.-   LUNDBERG, Jon O., et al. Nitrate, bacteria and human health. Nat Rev    Microbiol. 2004, no. 2, p. 593-602.-   MODIN, A., et al. Nitrite-derived nitric oxide: a possible mediator    of ‘acidic-metabolic’ vasodilation. Acta Physiol Scand. 2001, vol.    171, p. 9-16.-   COSBY, K., et al. Nitrite reduction to nitric oxide by    deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003,    no. 9, p. 1498-505.-   SPIEGELHALDER, B., et al. Influence of dietary nitrate on nitrite    content of human saliva: possible relevance to in vivo formation of    N-nitroso compounds. Food Cosmet Toxicol. 1976, no. 14, p. 545-548.-   LUNDBERG, Jon O., et al. Inorganic nitrate is a possible source for    systemic generation of nitric oxide. Free Radic Biol Med. 2004, vol.    37, p. 395-400.-   DURANSKI, M. R., et al. Cytoprotective effects of nitrite during in    vivo ischemia-reperfusion of the heart and liver. J Clin Invest.    2005, vol. 115, p. 1232-1240.-   GLADWIN, M. T., et al. The emerging biology of the nitrite anion.    Nat Chem Biol. 2005, no. 1, p. 308-14.-   LARSEN, F. J., et al. Effects of dietary nitrate on blood pressure    in healthy volunteers. N Engl J Med. 2006, vol. 355, p. 2792-3.-   BORG, G. Perceived exertion as an indicator of somatic stress. Scand    J Rehabil Med. 1970, vol. 2, no. 2, p. 92-8.-   ÅSTRAND, P-O, et al. Textbook in work physiology. McGraw-Hill, 1970.    ISBN 0070024065. p. 619.-   LUNDBERG, J O, et al. Inorganic nitrate is a possible source for    systemic generation of nitric oxide. Free Rad Bio Med. 2004, vol.    37, no. 3, p. 395-400.-   BROUWER, E. On simple formulae for calculating the heat expenditure    and the quantities of carbohydrate and fat oxidized in metabolism of    men and animals, from gaseous exchange (Oxygen intake and carbonic    acid output) and urine-N. Acta Physiol Pharmacol Neerl. 1957, no.    6, p. 795-802.-   GAESSER, G A, et al. Muscular efficiency during steady-rate    exercise: effect of speed and work rate. J Appl Physiol. 1975, no.    38, p. 1132-1139.-   LARSEN, F J, et al. Effects of dietary nitrate on blood pressure in    healthy volunteers. N Engl J Med. 2006, vol. 355, no. 26, p. 2792-3.

The invention claimed is:
 1. A method for decreasing whole body oxygenconsumption in a human subject with metabolic stress caused by acondition selected from the group consisting of: surgery, malnutrition,cancer, burn injury, traumatic injury, anorexia nervosa, thyroiddisorder, stress ulcer, sleep apnea, septic shock, insufficientperfusion of the intestines, and disturbed glucose homeostasis, themethod consisting of: orally administering to said human subject ananion of nitrate (NO₃ ⁻ ) in an effective amount that provides nitrate(NO₃ ⁻ ) in a dose of 0.01-1 mmole/kg body weight/24 h and in a formselected from the group consisting of: a liquid, chewing gum, tablet,lozenge, wafer, cake, and bar, wherein the administration results indecreased whole body oxygen consumption in said human subject.
 2. Amethod for decreasing whole body oxygen consumption in a human subjectwith metabolic stress caused by a condition selected from the groupconsisting of: surgery, malnutrition, cancer, burn injury, traumaticinjury, anorexia nervosa, thyroid disorder, stress ulcer, sleep apnea,septic shock, insufficient perfusion of the intestines, and disturbedglucose homeostasis, the method comprising orally administering to saidhuman subject a composition comprising two physiologically activepharmaceuticals, wherein one physiologically active pharmaceutical is ananion of nitrate (NO₃ ⁻ ) and the other physiologically activepharmaceutical is an anion of nitrite (NO₂ ⁻ ), wherein the anion ofnitrate (NO₃ ⁻ ) is from at least one plant and is in an effectiveamount that provides nitrate (NO₃ ⁻ ) in a dose of 0.01-1 mmole/kg bodyweight/24 h and the ratio of nitrate (NO₃ ⁻ ) to nitrite (NO₂ ⁻ ) isabout 5:1 to about 100:1 and wherein the administration results indecreased whole body oxygen consumption in said human subject.
 3. Themethod according to claim 2, wherein the ratio of nitrate (NO₃ ⁻ ) tonitrite (NO₂ ⁻ ) is about 5:1 to 30:1.
 4. The method according to claim1, wherein the salt of nitrate (NO₃ ⁻ ) is isolated from one or morenitrate-rich vegetables and/or fruits.
 5. The method according to claim2, wherein the ratio of nitrate (NO₃ ⁻ ) to nitrite (NO₂ ⁻ ) is about10:1.
 6. The method according to claim 1, wherein the salt of nitrate(NO₃ ⁻ ) is administered in the form of a lozenge.
 7. The methodaccording to claim 1, wherein the anion of nitrate is a sodium,potassium, calcium, zinc, arginine, or ammonium salt.
 8. The methodaccording to claim 2, wherein the anion of nitrite (NO₂ ⁻ ⁻) is asodium, potassium, calcium, zinc, arginine, or ammonium salt.
 9. Amethod for decreasing whole body oxygen consumption in a human subjectwith metabolic stress caused by a condition selected from the groupconsisting of: surgery, malnutrition, cancer, burn injury, traumaticinjury, anorexia nervosa, thyroid disorder, stress ulcer, sleep apnea,septic shock, insufficient perfusion of the intestines, and disturbedglucose homeostasis, the method consisting of orally administering tosaid human subject a composition consisting of: an anion of nitrate (NO₃⁻ ) in an effective amount that provides nitrate (NO₃ ⁻ ) in a dose of0.01-1 mmole/kg body weight/24 h; non-pathogenic live bacteria selectedfrom the group consisting of: probiotic bacteria, Veillonella species,Staphylococcus species, Actinomyces species, and Rothia species, and atleast one carrier, wherein the anion of nitrate (NO₃ ⁻ ) is from atleast one plant and wherein the composition reacts locally in thegastrointestinal tract via bacteria-dependent reduction of nitrate to NOand other bioactive nitrogen oxides and the administration of thecomposition results in decreased whole body oxygen consumption in saidhuman subject.
 10. A method for decreasing whole body oxygen consumptionin a human subject with metabolic stress caused by a condition selectedfrom the group consisting of: surgery, malnutrition, cancer, burninjury, traumatic injury, anorexia nervosa, thyroid disorder, stressulcer, sleep apnea, septic shock, insufficient perfusion of theintestines, and disturbed glucose homeostasis, the method consisting oforally administering to said human subject a composition consisting of:an anion of nitrate (NO₃ ⁻ ) in an effective amount that providesnitrate (NO₃ ⁻ ) in a dose of 0.01-1 mmole/kg body weight/24 h, and apolyphenol-rich compound or product, wherein the anion of nitrate (NO₃ ⁻) is from at least one plant and the administration of the compositionresults in decreased whole body oxygen consumption in said humansubject.
 11. A method for decreasing whole body oxygen consumption in ahuman subject with metabolic stress caused by a condition selected fromthe group consisting of: surgery, malnutrition, cancer, burn injury,traumatic injury, anorexia nervosa, thyroid disorder, stress ulcer,sleep apnea, septic shock, insufficient perfusion of the intestines, anddisturbed glucose homeostasis, the method comprising: administering tosaid human subject a composition comprising one physiologically activepharmaceutical, wherein the physiologically active pharmaceutical is anitrite (NO₂ ⁻ ), wherein the nitrite (NO₂ ⁻ ) is administered orally orintravenously in an effective amount that provides nitrite (NO₂ ⁻ ) in adose of 0.01-100 μmole/kg body weight and the administration results indecreased whole body oxygen consumption in said human subject.
 12. Themethod of claim 1, wherein the disturbed glucose homeostasis is acondition selected from the group consisting of: diabetes mellitus type1, diabetes mellitus type 2, drug-induced diabetes, metabolic syndrome,and obesity.
 13. The method of claim 11, wherein the disturbed glucosehomeostasis is a condition selected from the group consisting of:diabetes mellitus type 1, diabetes mellitus type 2, drug-induceddiabetes, metabolic syndrome, and obesity.
 14. The method according toclaim 2, wherein the composition further comprises ascorbic acid. 15.The method according to claim 2, wherein the composition has the form ofa liquid, chewing gum, tablet, lozenge, wafer, cake, or bar.
 16. Themethod according to claim 2, wherein the composition has the form of alozenge.
 17. The method according to claim 2, wherein the anion ofnitrate is a sodium, potassium, calcium, zinc, arginine, or ammoniumsalt.
 18. The method according to claim 2, wherein the compositionfurther comprises non-pathogenic live bacteria and reacts locally in thegastrointestinal tract via bacteria-dependent reduction of nitrate andnitrite to NO and other bioactive nitrogen oxides, wherein thenon-pathogenic live bacteria are selected from the group consisting of:probiotic bacteria, Veillonella species, Staphylococcus species,Actinomyces species, and Rothia species.